The proposed studies are designed to define the process of DNA-dependent RNA polymerase II-mediated gene transcription in the pathogenic protozoa, Trypanosoma brucei gambiense. The steps involved in this process and the molecules participating in each step will be examined in detail in an attempt to understand more fully the basic events of transcription and to lay the foundation for studies of the molecular basis for the selectivity and regulation of gene expression in this parasite. The RNA polymerase responsible for the transcription of genes which code for proteins (designated RNA polymerase II or B) will be isolated from trypanosomes and purified by ion-exchange chromatography and affinity chromatography. The enzyme will then be characterized physiochemically and the optimal reaction conditions for its activity will be determined. The purified enzyme will then be used to supplement cell-free extracts for the development of an in vitro transcription system with the following characteristics: 1) dependence upon exogenously added DNA; 2) the ability to specifically recognize genes with legitimate Class II promoters; 3) the capacity to support accurate initiation of transcription at the site(s) used for mRNA production in vivo. To facilitate the examination of this in vitro transcription system, defined segments of transcriptionally active tubulin genes will be used to assay the process. In addition, the structure of the early transcripts will be compared to these genes and to the sequence of the corresponding mRNAs. The cell-free extracts will then be fractionated and reconstituted systems will be assayed for transcriptional competence. The molecular components of the extracts which contribute to the process will be identified by physiochemical and immunological means employing electrophoretic techniques and monoclonal antibodies. These studies sh ould provide a clearer picture of this crucial cellular process in the trypanosomes and establish conditions under which more exotic gene families (such as the variant antigen genes) of the parasite can be studied in a directly manipulatable system in vitro. In addition, any differences in the process or the enzymes involved in it, may have potential use as targets for rational chemotherapy.